Current treatment of disease is predominantly non-targeted. Drugs are administered systemically, e.g. orally, which exposes many other tissues as well as the tissues which are diseased. In cancer therapy, chemotherapeutic drugs are specific for cells which are growing and dividing rapidly, as they work mainly by a mechanism which interferes with DNA replication [1]. However, other cells may take up the drug and also become intoxicated, such as rapidly dividing bone marrow stem cells, resulting in immunosupression. In infectious diseases, anti-bacterial drugs are introduced into the blood (orally or by injection) and interfere with a particular bacterial metabolic pathway. Again, exposure to other tissues can result in side effects. Virally-infected cells are difficult to treat as their metabolism is nearly identical to uninfected cells.
It is widely acknowledged that the future of medicine lies in the tailoring of drugs to the disease. This means delivering the therapeutic agent to the correct target tissue, rather than the non-selective hit and miss approach of most of the conventional drug treatments used today. This approach may result in the administration of lower doses, lower side effects and toxicities and overall better responses. Advances in genomics may one day mean that drugs can be tailored to the individual, as one individual's cancer may differ from another's.
There are many drugs used clinically today that are effective at destroying or treating diseased cells, once they have accumulated in the correct tissue. The problem therefore lies with the specific targeting of drugs, rather than the effector mechanism. Examples of targeting include targeted ionising radiation as opposed to external beam radiotherapy [2], targeted chemotherapy drugs (e.g. methotrexate or doxorubicin) as opposed to free drugs [3] and toxins [4]. Photodynamic therapy (PDT) is a particularly good example as it is already well established in many treatments. However, it is becoming apparent that a better therapeutic outcome may result from pre-targeting a photosensitizing (PS) drug to the correct tissues in addition to targeting the light source, which is not accurate at a cellular level [5].
Targeting drugs or other effectors to the desired cells has been previously proposed. One of the main approaches to targeting is to use antibodies as the targeting element of a multifunctional molecule [6]. The ideal design for such a multifunctional molecule would be one which is highly specific for diseased cells, able to carry many drugs with high capacity without compromising their function, and able to deposit the drug in the sub-cellular compartment which would primarily be affected.
Antibody Targeting
Antibodies have naturally evolved to act as the first line of defence in the mammalian immune system. They are complex glycoproteins which have exquisite diversity and specificity. This diversity arises from programmed gene shuffling and targeted mutagenesis, resulting in probably a trillion different antibody sequences [7]. Consequently, this diversity means that antibodies can bind to practically any target molecule. It is now possible to mimic antibody selection and production in vitro, selecting for recombinant human antibodies against virtually any desired target [8]. A significant number of biotechnological drugs in development are based on antibody targeting [6]. The most popular in vitro selection technique is antibody phage display, where antibodies are displayed and manipulated on the surface of viruses [8]. There are many therapeutic antibodies being developed for a range of diseases, primarily cancer. Table 1 lists some of these antibodies.
Antibodies can bind with a high degree of specificity to target cells expressing the appropriate receptor. The affinity of an antibody is a measure of how well an antibody binds to the target (antigen). It is usually described by an equilibrium dissociation constant (Kd). Technology exists to select and manipulate antibodies which have the desired kinetic binding properties. For antibodies that need to be internalised, the association rate is more important, as the dissociation rate does not function if the antibody is taken into the cell.
As with all biological molecules, the size of the antibody affects its pharmacokinetics in vivo [12]. Larger molecules persist longer in the circulation due to slow clearance (large glycoproteins are cleared through specific uptake by the liver). For whole antibodies (molecular weight approx. 150 KDa) which recognise a cancer cell antigen in a mouse model system, 30-40% can be taken up by the tumour, but because they persist longer in the circulation, it takes 1-2 days for a tumour:blood ratio of more than one to be reached. Typical tumour:blood ratios are 5-10 by about day 3 [13]. With smaller fragments of antibodies, which have been produced by in vitro techniques and recombinant DNA technology, the clearance from the circulation is faster (molecules smaller that about 50 KDa are excreted through the kidneys, as well as the liver). Single-chain Fvs (about 30 KDa) are artificial binding molecules derived from whole antibodies, but contain the minimal part required to recognise antigen [14]. Again in mouse model systems, scFvs can deliver 1-2% of the injected dose, but with tumour:blood ratios better than 25:1, with some tumour:organ ratios even higher [15]. As scFvs have only been developed over the last 10 years, there are not many examples in late clinical trials. From clinical trials of whole antibodies, the amount actually delivered to tumours is about 0.1 to 1% of that seen in mouse models, but with similar tumour:organ ratios [16]. If another molecule is attached to the antibody, then the new size determines the altered pharmacokinetic properties. Other properties such as net charge and hydrophilicity have effects on the targeting kinetics [17].
Some cell surface antigens are static or very slowly internalise when bound by a ligand such as an antibody. There are some which have a function that requires internalisation, such as cell signalling or uptake of metals and lipids. Antibodies can be used to deliver agents intracellularly. These agents can be therapeutic—repairing or destroying diseased cells. Examples include gene delivery [18], the intracellular delivery of toxins (e.g. Pseudomonas exotoxin [4]), enzymes (e.g. ribonuclease [19]) and drugs (e.g. methotrexate [3]). Some of these agents need targeting to particular sub-cellular organelles in order to exert their effects. Advances in cell biology have uncovered ‘codes’—amino acid sequences which direct intracellular proteins to certain sub-cellular compartments. There are specific sequences to target to the nucleus, endoplasmic reticulum, golgi, lysosomes and mitochondria (Table 2).
There has been much research into targetable therapeutic drugs where novel effector functions have been linked to antibodies or other targeting ligands. Some of these need to be internalised to successfully deliver a toxic agent. Many of these have shown good results in vitro and in vivo in animal models, but have been disappointing in the clinic. Immunotoxins have shown problems such as immune reactions and liver/kidney toxicity [25]. There have been developments with new ‘humanised’ immunotoxins based on enzymes such as ribonuclease [19] and deoxyribonuclease [26]. These potentially have lower side effects and are more tolerable, but still do not have a bystander killing effect. Chemotherapy drugs tend to be much less active when linked to proteins as they are not released effectively and radioimmunotherapy tends to irradiate other tissues en route to the tumour, giving rise to bone marrow and liver toxicity. Photosensitising (PS) drugs are particularly attractive agents to link to proteins, as the cytotoxic elements are the singlet oxygen species generated from them and not the PS drugs themselves [5].
Photodynamic Therapy (PDT)
Photodynamic therapy is a minimally invasive treatment for a range of conditions where diseased cells and tissues need to be removed [27]. Unlike ionising radiation, it can be administered repeatedly at the same site. Its use in cancer treatment is attractive because conventional modalities such as chemotherapy, radiotherapy or surgery do not preclude the use of PDT and vice versa. Photodynamic therapy is also finding other applications where specific cell populations must be destroyed, such as blood vessels (in age-related macular degeneration (AMD) or in cancer), the treatment of immune disorders, cardiovascular disease, and microbial infections. PDT is a two-step or binary process starting with the administration of the PS drug, by intravenous injection, or topical application for skin cancer. The physico-chemical nature of the drug causes it to be preferentially taken up by cancer cells or other target cells [28]. Once a favourable tumour (or other target): normal organ ratio is obtained, the second step is the activation of the PS drug with a specific dose of light, at a particular wavelength. This ultimately causes the conversion of molecular oxygen found in the cellular environment into reactive oxygen species (ROS) primarily singlet oxygen (1O2), although reactions of intermediate photochemically produced species also generate hydroxyl radicals (OH.) and superoxide (O2−.). These molecular species cause damage to cellular components such as DNA, proteins and lipids [29]. PDT is a cold photochemical reaction, i.e. the laser light used is not ionising and the PS drugs have very low systemic toxicity. The combination of PS drug and light result in low morbidity and insignificant functional disturbance and offers many advantages in the treatment of diseases. There is growing evidence that PDT response rates and durability of responses are as good as or even superior to standard locoregional therapies [27].
The light activation of ROS is highly cytotoxic. In fact some natural processes in the immune system utilise ROS as a way of destroying unwanted cells. These species have a short lifetime (<0.04 μs) and act over a short radius (<0.04 μm) from their point of origin. The destruction of cells leads to a necrotic area of tissue which eventually sloughs away or is resorbed. The remaining tissue heals naturally, usually without scarring. There is no tissue heating and connective tissue such as collagen and elastin are unaffected, resulting in less risk to the underlying structures compared to thermal laser techniques, surgery or external beam radiotherapy. More detailed research has shown that PDT induces apoptosis (non-inflammatory cell death), and the resulting necrosis (inflammatory cell lysis) seen is due to the mass of dying cells which are not cleared away by the immune system [30].
Generally PS drugs are administered systemically, with some topical applications for skin lesions. When the PS drug has accumulated in the target tissue, with ratios typically 2-5:1 compared with normal surrounding tissues (except in the brain where the ratio can be up to 50:1), low power light of a particular wavelength is directed onto the tumour (or the eye in AMD treatment [31]). Because human tissue can transmit light most effectively in the red region of the visible light spectrum, PS drugs which can absorb red light (630 nm or above) can be activated up to a depth of about 1 cm. Patients must avoid sunlight until systemically administered PS drugs clear from the body, otherwise they may have skin photosensitivity, resulting in skin burn.
The treatment scheme is attractive to the clinician in that superficial diseases can usually be treated with local anaesthesia and sedation. The generally low toxicity (with the possible exception of skin photosensitivity) limits the need for other medication. Topical treatments do not require sterile conditions and can be given in an outpatient clinic.
Research on a number of PS drugs including silicon phthalocyanines has shown that PDT induces apoptosis-programmed cell death [32]. Apoptosis is the highly orchestrated and evolutionary conserved form of cell death in which cells neatly commit suicide by chopping themselves into membrane-packaged pieces [33]. These apoptotic bodies are marked for phagocytosis by the immune system. Usually, too much apoptosis in a small area ‘overloads’ the immune system and the area eventually becomes necrotic, with inflammatory consequences.
Photofrin (porfimer sodium), 5-aminolaevulanic acid (ALA) and Verteporfin (BPD-benzoporphyrin derivative) are three PS drugs which have regulatory approval. A promising, potent second generation PS drug, Foscan (temoporfin; meta-tetrahydroxyphenyl chlorin) is encountering problems in acquiring approval from the FDA and MCA. Porfimer sodium, the first PS drug to be approved, is licensed for use in bladder, stomach, oesophagus, cervix and lung cancer. Its performance is moderate due to poor light absorption characteristics in the red end of the spectrum (activated at 630 nm), meaning it can only penetrate about 5 mm into tissues. It also persists in the body for weeks, leading to skin photosensitivity. However it has been effective in the treatment of the above cancers [27]. ALA is applied topically in the treatment of skin lesions and is converted endogenously to protoporphyrin IX, a naturally-occurring PS molecule. This can be activated at many wavelengths and its depth of effect is less than 2 mm. ‘Visudyne’ (Verteporfin) also performs well in AMD [31], without the issues of tissue penetration found in tumour applications.
The newer generation of PS drugs have longer activation wavelengths thus allowing deeper tissue penetration by red light, higher quantum yield and better pharmacokinetics in terms of tumour selectivity and residual skin photosensitivity. These classes of PS drugs include the phthalocyanines, chlorins, texaphyrins and purpurins. The synthetic chlorin, Foscan is a very potent PS drug with a wavelength of activation of 652 nm, good quantum yield of singlet oxygen and skin photosensitivity of about 2 weeks. There have been many clinical trials for a variety of cancers, with good results [27]. There are other PS drugs which have been developed and are in trials which can adsorb at 740 nm, such as meso-tetrahydrophenyl bacteriochlorin (m-THPBC).
Clinical PDT
PDT can achieve disease control rates similar to conventional techniques with lower morbidity rates, simplicity of use and improved functional and cosmetic outcome. PDT has mainly been used where conventional approaches have failed or are unsuitable. These include pre-malignant dysplastic lesions and non-invasive cancers which are commonly found in the mucosa of aerodigestive and urinary tracts (e.g. oral cavity, oesophagus and bladder). Current treatments for cancer at this stage are not very successful and good responses here would prevent larger solid tumours or metastatic spreads occurring. Treatment for Barrett's oesophagus usually involves an oesophagectomy, which requires general anaesthesia, has a risk of morbidity and loss of function and disfigurement. PDT is being seen as an attractive option because of the large area which can be treated superficially with less risk. Photofrin, ALA and Foscan have produced good responses in these types of cancers in clinical trials (Table 3).
Due to easy light accessibility, the treatment of cutaneous disease such as skin cancer has produced good results with systemic and topical PS drugs (Table 3). Head, neck and oral lesions have also produced good results and are well suited due to the good cosmetic outcome of the treatment (Table 3). Treatment of other cancers are being tested as advances are being made in laser and light delivery technology. Endoscopes can be used to deliver the activating light dose to any hollow structure such as the oesophagus and bronchial cavity, thus expanding the treatment range to gastrointestinal and lung cancers (Table 3) with minimal surgery. Large areas such as the pleura and peritoneum can be treated, where radiotherapy would not be able to give a high enough curative dose. PDT has great promise in the treatment of these surface serosal cancers, in combination with debulking surgery. Light can be delivered to these large surfaces in a short time, through hollow cavities. The limited depth of activity would be an advantage, as the critical underlying organs would be spared (Table 3). Adjuvant therapy is also an option being investigated, where the solid tumour is surgically removed and any remaining tumour cells are destroyed by one round of PDT in the cavity formed.
Although surface cancers may be the most amenable to PDT, solid tumours may also be able to undergo interstitial treatment, where the PS drug is administered systemically or by intra-tumour injection, followed by the insertion of laser fibres through needles equally spread throughout the tumour. This can result in necrosis of very large tumours (Table 3).
To summarise, there are several advantages of PDT therapy. It offers non-invasive, low toxicity treatments which can be targeted by the light activation. The target cells cannot develop resistance to the cytotoxic species (ROS). Following treatment, little tissue scarring exists. However, PS drugs are not very selective for the target cells with target:blood ratios typically in single figures. Because PS drugs “piggy-back” on blood proteins, they persist longer in the circulation than is desired, leaving the patient photosensitive for 2 weeks in the best of cases. It is becoming increasingly clear that PS drugs need to accumulate inside cells as the generated ROS have a short pathlength. This may not be achieved effectively with current PS drugs.
Targetable PDT
Photosensitiser drugs can still be active and functional while attached to carriers, as the cytotoxic effect is a secondary effect resulting from light activation. This makes them very amenable to specific drug delivery mechanisms. Currently, the approaches used to link PS drugs to targetable elements include direct conjugation of derivatised PS drugs to whole monoclonal antibodies or other ligands [34-37]. However, this often results in a heterogeneous mixture of antibody-PS drug molecules as the chemistry is not accurate. Whole antibodies have a molecular weight of 150 KDa, resulting in very large immunoconjugates with unfavourable pharmacokinetics, such as poor tumour:organ ratios [36] which take a long time to achieve. It is also likely that PS drugs linked to large adjacent residues of a protein can have a detrimental effect on PS photophysics, with quenching of the desired PS excited states occurring due to adverse PS-protein interactions. The non-specific attachment of PS drugs onto antibodies or other ligands can result in a severe compromise in binding ability of the ligand. The antibody binding site may be hindered by such reactions, dramatically lowering the affinity and specificity of the antibody. Too many PS drugs attached can also affect the hydrophobicity of a protein and may have an adverse effect on the structure and pharmacokinetics [36].
Some researchers have tried to circumvent these problems by attempting to link PS drugs to designated ‘carriers’ such as chemically synthesised branched carbohydrate chains and poly-lysine chains. These approaches all require additional conjugation steps as the ligand-carriers cannot be made entirely recombinantly. Using chains of pure poly-lysine may also give rise to problems, for example, proteolyic instability in vivo, or the concentration of hydrophobic PS drugs in one part of the molecule leading to aggregation and quenching of adjacent PS drugs-excited states.
The present invention seeks to alleviate some of the above-mentioned problems of the prior art, thereby providing an improved system for targeting and delivery of therapeutic and/or diagnostic agents.